Process for producing lincomycin

ABSTRACT

A NOVEL MICROBIOLOGICAL PROCESS FOR PREPARING THE ANTIBIOTIC LINCOMYCIN.

' U.S. Cl. 195-80 R United States Patent Oflice 3,726,766 Patented Apr. 10, 1973 3,726,766 PROCESS FOR PRODUCING LINCOMYCIN Alexander D. Argoudelis, Portage, and John H. Coats,

Kalamazoo, Mich., assignors to The Upjohn Company,

Kalamazoo, Mich.

No Drawing. Filed June 28, 1971, Ser. No. 157,729 Int. Cl. C12d 9/20 3 Claims ABSTRACT OF THE DISCLOSURE A novel microbiological process for preparing the antibiotic lincomycin.

BRIEF SUMMARY OF THE INVENTION Lincomycin is an antibiotic producible by a fermentation process using the microorganism Streptomyces lincolnensis var. lincolnensis. In U.S. Pat. 3,086,912 there is described a fermentation and recovery process for the production of lincomycin, formerly known as lincolnensin. The microbiological process of the subject invention comprises the use of a novel microorganism to produce lincomycin.

DETAILED DESCRIPTION OF THE INVENTION The novel actinomycete used according to this invention for the production of lincomycin is Streptomyces pseudogriseo lus chemovar linmyceticus Dietz var. nova. One of its strain characteristics is the production of lincomycin. A subculture of this living organism can be obtained upon request from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Services, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number of this repository is 'NRRL 3985.

DESCRIPTION OF THE MICROORGANISM Streptomyces pseudogriseolus chemovar linmycen'cus Dietz var. nova Color characteristics.Aerial growth predominantly gray. On a few media aerial growth may be red, white, or gray-white. Melanin-negative. Appearance on Ektachrome, as described in Dietz [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. NY. Acad. Sci. 60:152-154; Dietz, A., and I. Mathews, 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscqpi'cus. Appl. Microbiol. 10:258-263; Dietz, A. 1967. Strept0myces stefiisburgents sp. n. J. Bacteriol. 94:2022-2026] is given in Table I. Reference color characteristics are given in Table II. The culture may be placed in the Gray (GY), White (W), and Red (R) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1962. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338].

Microspic characteristics.Sporophores short to moderate in length, appearing as straight (RF) to open spiral (RA) to spiral (S) in the sense of Pridham, et al. [Pridham, T. G. and -D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an acid for species determination. J. Bacteriol. 56:107- 114]. Spores, examined by the Transmission Electron Microscope (TEM) procedures of Dietz and Mathews [Dietz, A. and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscopicus. Appl. Microbiol. 10:258463; Dietz A. and J. Mathews. 1970. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533], are oval to oblong with short spines on the surface. Spines are sparse.

Cultural characteristics-See Table III.

Carbon utilization.--Growth of the culture on carbon compounds was determined using the synthetic medium of Pridham Gottlieb [Pridham, T. G. and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107-114], Table IV, and their modified medium [Shirling, E. B. and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 162313-340], Table V.

Temperature.--The culture grew at temperatures of 18 C. to 55 C. on Bennetts, Czapeks sucrose, and maltose-tryptone agars. Growth at 18 C. and 55 C. was predominantly vegetative. There was slight aerial growth at 24 C. Good aerial and vegetative growth occurred at 28 C. to 45 C.

Antibiotic-producing properties-Streptomyces pseudogriseolus ATCC 12770, produces the antibiotic antitoxoplasmic substance No. 534 (xanthomycin) [Ok'ami, Yoshiro, R. Utahara, H. Oyagi, S. Nakamura, and H. Umezawa. 1955. The screening of anti-toxoplasmic substance produced by streptomycete and anti-toxoplasmic substance No. 534. The journal of Antibiotics, Ser. A 8:126-128]. Streptomyces pseudogriseolus chemovar linmyceticus produces the antibiotic lincomycin.

Streptomyces pseudogrideolus chemovar linmyceticus is a new soil isolate of the genus Streptomyces.

A distinctive property of the culture is the production of the antibiotic lincomycin. This culture is readily distinguished from the lincomycin-producing cultures Streptomyces lincolnensis var. lincolnensis [Mason, D. 1., A. Dietz, and C. De Boer, 1962. Lincomycin, a new antibiotic. I. Discovery and Biological Properties. Antimicrobial Agents and Chemotherapyl962, pp. 554-559] and Streptomyces espz'nosus (application Ser. No. 102,- 141, filed Dec. 28, 1970) by color pattern, sporophore, and spore type. S. lincolnensis has pale pink aerial growth,

is melanin-positive and has rectangular smooth spores borne in long straight to fiexnous sporophores. S. espznosus has gray-green aerial growth, is melanin-negative and has round spiny spores borne in short straight to open-spiral sporophores. S. pseudogriseolus chemovar linmyceticus has gray aerial growth, is melanin-negative, and has oval to oblong sparsely spiny spores borne in short to moderately long straight to open spiral to spiral sporophores.

The new lincomycin-produciug culture is very similar to the type culture Streptomyces pseudogriseolus Okami and Umezawa, ATCC 12770. The cultures differ slightly in color pattern, utilization of some carbon compounds, and in antibiotic-production. The significant differentiat- Washington, DC.

ing characteristics of the new soil isolate, from the type TABLE I culture S. pseudogriseolus, is the production of the chemi- Appearance of S. pseudogriseolus cultures on Ektachrorne cal entity the antibiotic lincomycin. According to Rule 8, Recommendation -8a of the International Code of s-pmdoflriseolus Nomenclature of Bacteria [International Code of Nomen- Agar medium iiiii ft iitus i r il i ii clature of Bacteria, 1966. Edited by the editorial board of the Judicial Commission of the International Committee Gray on Nomenclature of Bacteria. Intern. J. System. Bacteriol. Tan (With red t g 16:459-490], the term chemovar may be used to desig- Gray Gra,y

mate a strain producing some chemical not normally pro- Red'gl'ay Gray (with fed-tinge)- duced by the type strain of the species. Gray Gray,

The characteristics of Streptomyces pseudogriseolus Pep Es Deep red-brown Light red-brownchemovar linmyceticus Dietz var. nova., NRRL 3985, are No m i growth NO with growth given in the following tables: Yellow-ta" Yellow-tan- Table I.-Appearance of S. pseudogriseolous cultures on Traee gray Trace gray. Ektachrome. SEES-Larch Rad Table IL-Reference color characteristics of S. pseus Gray Gray.

R Very pale red-gray Recl gray.

dogriseolus cultures.

Table III.General cultural characteristics of S. pseudogriseolus cultures.

Table IV.-Utilization of carbon compounds in the synthetic medium of Pridham and Gottlieb.

Table V.--Utilization of carbon compounds in the modified medium of Pridham and Gottlieb.

Norm-S =Suriace; R Reverse.

TABLE II Reference color characteristics of S. pseudogriaeolus cultures Dietz, A., Ektachrorne Transparencies as Aids in Actinomycete Classification", Annals of the New York Academy of Sciences, :152-

Color Harmony Manual, 3rd ed. 1948 NBS Circular 553 Y S. pseudogriseolus chemovar g7gigeudogrz'seolus ATCC S. pseudogriseolus chemovar S. pseudogrireulus ATCC 12770 Agar medium linmycetieus linmycetieus Bennett's:

s e gray 5 ie ashes 63 In light brownish gray, R 2 ge covert tan, gricge 3 ig beige brown, mist 94 in light olive brown; m grayish yellowish brown. 109 gm light grayish brown; g moderate olive. olive brown.

ora eli'uioei T n e gray 3 to silver gray 63 gm light brownish gray. R e gray 3 lb silver gray Do. 8... Maltose rypton s e gray 0 light gray 264 gm light gray. R 3 ig beige brown, mist 2 ie light mustard tan 80 m grayish yellowish 91 gm dark grayish yellow;

brown. brown; 95 g moderate 94 g light olive brown;

olive brown. 106 g light olive. P 2 li gm grayish olive Yeast extract-malt extract (lSP-2) e gray 5fe ashes. R 3 li beaver 4 li beaver 80 m gayish yellowish brown; 95 g moderate olive brown.

Oatmeal USP-3):

S 4 ge light fawn, rose beige... 3 to silver gray 29 in moderate yellowish pink; 57 g light brown. R 4 ge light fawn, rose beige.-. 3 go beige ,camel.. 29 moderate yellowish pink; 57 g light brown; P Inorganic-salts starch (SP-4):

S C light gray 3 to silver gray 264 gm light gray R 3 go beige, camel to 6 li 5 ih lead gray, shadow gray. 76 gm light yellowish dark rose taupe. gray. brown to. P 5 ig (around growth rose 3 go beige camel 45 in light grayish reddish taupe. brown; 46 g grayish reddish brown; 61 g grayish brown. Glycerol asparagiiie (lSP-fi): Y

S 5 ob 3 ml beaver gray to 3 le silver gray.

1t 3 go light taii 3 li beaver to 1 dc putty, 76 gm light yellowish griege. b

63 n1 light brownish gray.

61 m grayish brown; 81 g dark grayish yellowish brown.

63 gm light brownish gray.

76 gm light yellowish brown.

96 g dark olive brown; 266 m dark gray-to 63 gm light brownish giay.

80 m grayish yellowish brown; 95 g moderate olive brown to; 121 iii pale yellow green; 122 g grayish yellow green.

1 Jacobson, E., W. Granville, and C. E. Foss. 1948. Color harmony manual, 3rd ed. Container Corporation of America, Chica o.

C. 2 Kelly, K. L.and D.

N0iE.-S Surface; R Reverse; P Pigment.

E B. Judd. 1955. The ISCC-NBS method of designating colors and a dictionary of color names. U.S. Dept. of Comm. Circ. 553,

TABLE III General cultural characteristics of S. pseudogriseolus cultures 8. preudogriseolus chernovar linmyceticus S. pseudogriseolus ATCC 12770 Agai meidia I:

oneron: S Trace white aerial growth Pale gray-white aerial growth.

R Yellowcrcam Yellow-tan. P Yellow Yellow. 0 Melanin negative Melanin negative. Calcium malate:

S No aerial growth Poor gray-tan aerial growth.

...................... Co1orless. Gray-tan.

.. None None.

. Malate not solubilized Malate not solubilized.

Gray-white aerial growth. Gray-yellow. Pale pink-tan.

Trace gray-white aerial growth. Yellow-orange.

Do. Casein solubilized under growth.

Heavy gray aerial growth. Maroon.

Red-tan.

Tyrosine solubilized.

Pale gray aerial growth ed t Xanthine:

S Pale gray aerial grow h Heavy gray aerial growth. R Olive-cream.. Olive-gray-cream. P Pale cr Pale olive. O Xanthine solubihze Xanthine solubilized. Nutrient starch:

S Pale gray aerial grow h Heavy gray aerial gro th. R Pale red-tan Olive-gray-cream. P ..do Pale olive. 0 Starch hydrolyzed Starch hydrolyzed. Yeast extract-malt extract:

S Gray-white aerial gl'OWth Heavy gray-white aerial growth. R Maroon Red-tan to red-cream on edge. I Light red-tan Red-tan. Tubed media Gelatin Plain S Colorless surface growth i G ray aerial growth. P None Red pigment in top diffused in boti om O Liquefaction complete in four tubes; partial in Liquefaction two. Nutrient S Colorless surface growth Colorless surface growth. P None to pale tan None. 0 Liquefaction complete Liquefaction in four tubes; complete in two. Litmus milk S Heavy surface DQ111018 With blue tinge and trace Heavy surface pellicle with blue tinge and trace gray aerial growth. gray aerial growth. 0 Partial peptonization; Partial decolorization; Partial peptonization; Complete decoloiization;

Partial coagulation; pH 6.9. pH 7.2. Nitrate broth Synthetic S Pelleted COIOrIeSS surface ri g Pclleted colorless surface ring.

Nonc. None. Growth throughout broth; Flocculcnt growth Growth throughout broth; Flocculent growth at at base; Nitrate not reduced to nitrite. base; Nitrate not reduced to nitrite.

Heavy colorless surface ring with trace white Good Surface growth with trace white aerial aerial grow growth.

None None.

Heavy fiocculent growth at base; Nitrate not Heavy flocculent growth at base; Nitrate not reduced to nitrite. reduced to nitrite.

No aerial growth Trace white aerial growth. Colorless to yellow-tan Yellow-tan. None to pale yellow-tan Yellow-tan. Melanin-negative... Melanin-negative.

Good gray aerial growth. Gray.

None. Melanin-negative.

No aerial growth 1 In petridishes. N o'rE.-S Surface; R= Reverse; P Pigment; O Other characteristics.

T 313 Iv TABLE IVContinuccl Utilization of carbon compounds in the synthetic medium of Pridharn and Gomieb 1 6O pseudoqrz'seolus S.

chemovar pseudoqriseolus S. pseudoqriseolm linrnyceticus ATCC 12770 chemovar pseudoqrz'seolus linmyceticus aroc 12770 L-arabinose E Rhamnose D-fructose D-galactose 5f Na acetate Na citrate :1: Na succinate 1 Pridharn, 'r. (3., and D. Gottlieb. 1946. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. 51*) J. Bacteriol. 56:107-114. N0'rE.+=Positive utilization; -=Negative utilization; (-)=Slight TAB LE V Utilization of carbon compounds in the modified medium of Pridham and Gottlieb 1 Cellulose l Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:3l3340.

2 No growth.

3 Good growth.

NOTE.++=Strng utilization-Growth on tested carbon in basal medium is equal to or greater than growth on basal medium plus glucose; -=Utilization negative-Growth is similar to or less than on basal medium without carbon.

Lincomycin is produced by the novel microorganism of the subject invention when said microorganism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include corn steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, usually need not be added to the fermentation media since tap water and unpurified ingredients are used as media components.

Production of lincomycin by the process of the invention can be effected at any temperature conducive to satisfactory growth of the novel microorganism, for example, between about 18 and 40 C., and preferably between about 20 and 32 C. Ordinarily, optimum production of lincomycin is obtained in about 2 to days. The medium normally remains basic during the fermenta tion. The final pH is dependent, in part, on the buflers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avid a pronounced lag in the production of lincomycin and the attendant ineflicient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetaive inoculum is produced can be the same as, or different from, that utilized for the production of lincomycin, as long as it is such that a good growth of the microorganism is obtained.

The microorganism of the subject invention can also be grown in the media and under the conditions disclosed in U.S. Pat. 3,086,912. Further, the lincomycin compound produced by the subject process can be recovered by the procedures disclosed in U.S. 3,086,912.

In a preferred recovery process, lincomycin is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration and centrifugation. Lincomycin is then recovered from the filtered or centrifuged broth by extraction with a water-immiscible organic solvent in which lincomycin is soluble, for example, l-butanol, methyl ethyl ketone, benzene, and methylene chloride (preferred). Advantageously, the extraction is carried on after the filtered fermentation beer is adjusted to a pH of about 8.5 to 10.0 with a base, for example, sodium hydroxide. The solvent extract containing lincomycin can be concentrated to an oily material, which can then be subjected to extraction with ether and acidic methanol to give a colorless amorphous preparation of the acid salt of lincomycin.

It is to be understood that the process of the subject invention, though described in detail with particular reference to the novel microorganism Streptomyces pseudogriseolus chemovar linmyceticus Dietz War. nova, NRRL 3985, is not limited to this particular microorganism or to microorganisms fully described by the cultural characteristics disclosed herein. It is intended that this invention also include other strains or mutants of the said microorganism which can be produced by procedures well known in the art, for example, by subjecting the novel microorganism to X-ray or ultraviolet radiation, nitrogen mustard, phage exposure, and the like.

Hereinafter are described non-limiting examples of the process of the present invention. All percentages are by weight and all solvent mixture portions are by volume unless otherwise noted.

EXAMPLE 1 (A) Fermentation A soil slant of Streptomyces pseudogriseolus chemovar linmyceticus Dietz var. nova, NRRL 3985, is used to inoculate a series of SOD-ml. Erlenmeyer flasks each containing ml. of seed medium consisting of the following ingredients:

Glucose monohydrate 25 Pharmamedia 25 Tap water, q.s Balance *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Tex.

The flasks are grown for 3 days at 28 C., on a rotary shaker.

Five percent of the seed inoculum, described above, is used to inoculate a 500-ml. Erlenmeyer fermentation flask containing 100-ml. of sterile medium consisting of the following ingredients:

Lactose 10 Phytone* 10 NaCl 5 NaNO ,1 CaCO l0 Tap water, q.s. Balance *A plant peptone supplied by B.B.L., Division of Bio-Qucst, Cockeysville, Md.

bacterial spectrum as determined by a standard microbiological disc plate assay.

(B) Extraction Whole fermentation broth is filtered using diatomaceous earth as a filter aid. The filter cake is washed with 2 1. of water and the wash is combined with the clear filtrate (approximately 9 1., pH 8.8). The clear filtered wash is then mixed with potassium carbonate (200 g./l.). The pH is approximately 11.8. The alkaline solution is then extracted three times with 3 1. of methylene chloride each time. The methylene chloride extracts are combined and this solution is concentrated to dryness. The resulting residue is then dissolved in 500 ml. of ether. Methanolic hydrogen chloride (1 N, 5 ml.) is added to the ether solution to precipitate lincomycin hydrochloride which is then isolated by filtration and dried; yield, 200 mg. This preparation is characterized by thin layer chromatography 10 using silica gel G (Merck A.G., Darmstadt) as a support and methyl ethyl ketone-acetone-water (18625220 v./v.) as the solvent. The preparation is also characterized by using infrared and nuclear magnetic resonance spectroscopy. All of these characterization tests identify lincomycin, a well known antibiotic, in the preparation.

What is claimed is:

1. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces pseudogriseolus chemovar linmyceticus Dietz var. nova, having the identifying characteristics of NRRL 3985, and lincomycin producing mutants thereof, in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of lincomycin.

2. A process, according to claim 1, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.

3. A process, according to claim 1, wherein said lincomycin is isolated from the fermentation broth.

References Cited UNITED STATES PATENTS 3,086,912 4/1963 Bergy et al 80 X A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner 

